Dextranase (1,6-.alpha.-D-glucan 6-glucanohydrolase; EC 3.2.1.11) is an enzyme which hydrolyzes dextran. Dextran is a polymer of .alpha.-D glucopyranoses predominantly linked by 1,6 glycosidic bonds.
The initial interest in dextranase was its application in the commercial production of clinical dextran, used as blood substitute or extender (Tam, S. et al., 1976, Proc. Natl. Acad. Sci. USA. 73: 2128). Because of its ability to hydrolyze dextran, dextranase has also been found highly useful for hydrolyzing the dextran produced by the action of microorganisms causing dental caries or tooth decay. Thus, the incorporation of this enzyme into toothpaste or other oral detergent is considered in the art to provide a preventive or curative for tooth decay. See for example, the U.S. Pat. Nos. 3,991,177; 4,115,546; 4,140,758; 4,335,101; 4,438,093; 4,466,954; 4,469,673; 5,000,939; and 5,145,665.
In addition, dextranase is used in the sugar industry. Dextran is not a natural constituent of sugar juice, but it is formed by bacteria of the Leuconostoc species, which grow in damaged cane, and in juices that are delayed before processing through the evaporators. Dextran is a polymer which exhibits a high viscosity in solution. This increased viscosity becomes troublesome to both clarification and evaporation of juices and to the concentration of molasses when the level of dextran approaches 1000 parts per million of solid juices. Industrial dextranases are used to degrade the polymer to smaller molecules that do not create excessive viscosity. At a cane sugar refinery, dextran containing juice shows not only low filtration rate and poor crystallization of sucrose but also is the cause of serious quality problems of the final product.
Dextran is also formed occasionally in the processing of sugar beets, particularly when they have been exposed to freezing and thawing cycles during harvest and storage. The dextranase as described for cane sugar can be applied when this occurs.
The use of dextranase to hydrolyze dextran found in cane juices allows to increase the yield in sugar production (Imrie, F. K. E. et al., 1972, Sugar Technol. Rev. 1: 291-361; Fulcher, R. P. and Inkerman, P. A., 1976, Proc. Queensl. Soc. Sugar Cane Technol. 43: 295-305; Inkerman, P. A. and James, G., 1976, Proc. Queensl. Soc. Sugar Cane Technol. 43: 307-320; Inkerman, P. A. and Riddell, L., 1977, Proc. Queensl. Soc. Sugar Cane Technol. 44: 215-223; Inkerman, P. A., 1980, Proc. Int. Soc. Sugar Cane Technol. 17: 2411-2427; Barfoed, S. and Mollgaard, A., 1987, Zuckerindustrie (Berlin) 112(5): 391-395; Brown, C. F. and Inkerman, P. A., 1992, J. Agric. Food Chem. 40(2): 227-233).
Several microorganisms are capable of producing dextranase, among them fungi of the Penicillium, the Aspergillus, the Fusarium and the Chaetomium genera; Lactobacillus, Streptococcus and Flavobacterium bacteria, and yeasts such as the Lipomyces starkeyi, see for example, U.S. Pat. Nos. 2,742,399; 3,663,371; 3,875,009; 3,912,594; 4,732,854 and 4,820,640.
Commercial production of dextranase is by fermentation with either Penicillium sp. (Novo, 1977, Dextranase Novo 25L. A dextran decomposing enzyme for the sugar industry. Product data information 112-GB, Novo Enzyme Division, Hagsvaerd, Denmark) or Chaetomium sp. (Miles, 1986, Dextranex. Fungal dextranase for the sugar cane industry. Technical information L-1475, Miles Laboratories, Inc. Elkhart, Ind., USA). However, none of these fungi constitute an enzymatic source accepted by the U.S. Food and Drug Administration (FDA) (Koenig, D. W. and Day, D. F., 1989, Applied and Environmental Microbiology 55(8): 2079-2081).
Yeast, meanwhile, is a microorganism widely used in the food industry and most recently, in the production of pharmaceutical products. Yeast can also grow to a high cellular density in less time than fungi and it is therefore an appropriate host for industrial production of secreted proteins.
Only three groups have reported the cloning of genes coding for dextranases from different bacteria (Barrett, J. F. et al., 1987, Infection and Immunity 55(3): 792-802; Lawman, P. et al., 1991, Journal of Bacteriology 173(23): 7423-7428 and Okushima, M. et al., 1991, Jpn. J. Genet. 66: 173-187) and of these, only one nucleotide sequence has been published (Okushima, M. et al., 1991, Jpn. J. Genet. 66: 173-187). Neither reports about molecular cloning or sequencing of any fungal gene coding for dextranase nor works about the heterologous expression of dextranase in yeast have been published.